INA CTIVA TION- OF ENZ YMIE-SUBSTRA TE FILMS B Y SMALL DOSES OF X-RA YS - PNAS
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328 GENETICS: MAZIA AND BLUMENTHAL PRoc. N. A. S.
16 Slater, E. C., Nature (London), 161, 405-406 (1948).
17 Schonberg, A., Moubasher, R., and Mostafa, A., J. Chem. Soc. (London), 1948,
176-182.
INA CTIVA TION- OF ENZ YMIE-SUBSTRA TE FILMS B Y SMALL
DOSES OF X-RA YS
BY DANIEL MAZIA* AND GERTRUDE BLUMENTHAL
DEPARTMENT OF ZOOLOGY, UNIVERSITY OF MISSOURI
Communicated by L. J. Stadler, May 17, 1948
The analysis of the biological effects of radiations in terms of their effects
on simpler biochemical systems is rendered difficult by the fact that most
biocliemical systems seem so insensitive to radiations. In particular, chro-
mosomes are often affected, structurally and genetically, by doses smaller
by orders of magnitude than those which affect enzyme solutions, virus prep-
arations, etc., measurably. This apparent paradox contaitis certain clues
for the further analysis of biological effects. It focuses attention on the
chromosomes as the sensitive agents in the cell. This apparent sensitivity
is based in part on method. Chromosomal effects are often expressed as
visible changes, so that a single efect may be detected. The genetic units
in the chromosome are unique, so that the consequences of a single event at
the molecular level can be far-reaching and persistent. This contrasts with
the statistical nature of most chemical measurements.
But the question of the radiation sensitivity of chromosomes involves
factors not represented in an enzyme or virus solution, most clearly evident
where a single radiation event produces a break in a microscopic structure.
The chromosome is a continuous "solid" body, varying in both structure
and sensitivity through its own cycle and according to experimental condi-
tions: In attempting to visualize the effects of radiation, the structural fac-
tor has to be introduced. Therefore it would be desirable to set up a bio-
chemical system in which the factor of intermolecular structure was present
and in which the variables encountered by the chromosomes could be
tested. If such a system showed a high radiation sensitivity, this would in
itself suggest a relation between structure and sensitivity.
We have, in accordance with these requirements, investigated the radia-
tion sensitivity of surface films containing a proteolytic enzyme and its sub-
strate. Mazia, Hayashi and Yudowitchl have described the structure and
enzymatic activity of such a system involving pepsin and albumin, and have
considered possible parallels with chromosome structure and activity. Pre-
liminary experiments showed that this system was indeed sensitive to radia-
tions. X-ray doses of the 50-150 r order produced extensive inactivation.
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The sensitivity seemed to depend on the structural configuration, for the
same doses applied to the solutions from which the films were prepared had
no detectable effect. The sensitivity might depend on alterations of the
molecules on spreading, on the participation of sensitive intermolecular
bonds, not present in the solution, in the reaction, or merely on the geom-
etry of the exposure of the films to the irradiated medium. - All of these
possibilities may also be implicated in the sensitivity of chromosomes, and
all of them would be absent in experiments on solutions. The present series
of investigations is an exploration of these structural factors in radiation
sensitivity. The possible analogies between the actual structure of the
films and of chromosomes are not essential to such an exploration.
Methods.-Each experiment consists of the following steps. (1) A film
containing both enzyme and substrate is prepared by spreading at a pH at
which the activity of the enzyme is negligible. (2) The film is irradiated.
(3) The film is compressed into a fiber which is removed and washed.
(4) The enzymatic activity in the fiber is estimated by bringing the fiber
to the pH optimum of the enzyme and timing its self-digestion as observed
microscopically.
Solutions.-The staiting solutions consisted of mixtures of crystalline egg
albumin, prepared by the method of LaRosa,2 and crystalline pepsin (Lehn
and Fink Co.) in water. The albumin was dialyzed in the cold and frozen
dried. As this treatment produced pepsin preparations of varying activity,
we used the pepsin-MgSO4 mixture without further purification, estimating
its protein content by the niethod of Robinson and Hogden.3 The total
protein concentration used was 0.2 per cent. A small amount of isopropyl
alcohol was used to improve spreading.
Preparation of Films.-The films were spread over 10-fold diluted Mac-
Ilvaine buffer, the final pH being 4.0, in a Cenco Hydrophil tray. Pyrex-
redistilled water was used. The protein solution was measured onto the
previously swept surface with a 0.1 ml. pipette. In most of the experi-
ments, 0.05 ml. of the solution, a slight excess over what would be required
to cover the surface, under ideal conditions, was used. This meant that
there was a small amount of surplus protein, about 0.03 mg. of albumin and
about 0.001 mg. of pepsin, dissolved in the liter or so of buffer under the
film. The buffer was changed for each experiment, so that there would be
no accumulation of dissolved protein. The possibility of misinterpretation
of the results because of the presence of dissolved enzyme will be considered
later.
Irradilation.-The film trough was placed under the x-ray tube so that the
target was directly over the center of the-film. The standard area irradiated
was 18 X 32 cm. The dose rate was controlled by varying the target-to-
film distance, but, to insure covering the film, this was never less than 20 cm.
The Coolidge tube was operated at 150 kv. and 4 amperes with a 1-mm.
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Al filter. Dose was not measured for each experiment, but was calculated
from calibrations made with a Victoreen dosimeter set at various points in
the film tray, the tray being filled with paraffin. By this means, allowance
could be made for back-radiation, and for variations inherent in the geom-
etry of the system. Doses given in the data below are those immediately
below the target, and therefore represent the maximum received by the
films.
Test of Activity.-The activity of pepsin-albumin films may be detected
and measured microscopically. The film is compressed into a fiber by
bringing the barriers together, and the fiber is removed and washed as free
as possible of solutes from the trough with distilled water. The washed
fiber is then placed in HCl at pH 1.5 and observed microscopically. Fibers
containing active pepsin are observed to digest themselves and disappear
rapidly. Control fibers containing albumin and pepsin in a 40 to 1 ratio
digest in 30 to 50 seconds at pH 1.5 and 25°C. Mazia and Hayashi4 have
tested this microscopic method by comparison with measurements on the
rate of liberation, under similar conditions, of trichloracetic-soluble split
products, following the methods of Anson.5 The results have invariably
been parallel; the slower the disappearance of the fibers, the lower is the
rate of release of split products. While the time required for visible diges-
tion may not be a precise measure of the rate of digestion, we have never en-
countered a case where visible digestion was'not accompanied by release of
split products or where a fiber that did not visibly digest gave a full.yield of
split products. We consider, therefore, tat a slowing down or complete
inhibition of visible digestion is a measure of inactivation, and this was the
measure used in testing the effects of irradiation. For the present, our
chemical method is not suitable for the type of experiment described in this
paper, since it requires about 1 mg. of dried fiber.
Results.-Rate of Digestion as a function of x-ray Dose.-The effects of
radiation on digestion-time are shown in figure 1. The two curves represent.
films prepared from solutions containing pepsin-albumin ratios of 20:1 and
40:1. Each point represents a completely independent experiment. We
shall not at this time consider the source of variability in the observations,
except to mention that later work indicates that sensitivity is very depend-
ent on surface pressure which was not controlled in the experiments shown
in figure 1. The trend of the results is clear enou7gh. Effects of doses of the
order of 50 r may be detected and doses over 150 r produce the maximum
effect that can be observed: complete failure of the compressed film to di-
gest. The results suggest very strongly a "threshold" effect. In terms of
delay in digestion time, doses below 50 r are not veiy effective compared
with the same increments of dose above 50 r.
AMeasurement of Effect.-While the results given do show that we are
dealing with a highly radiosensitive- enzyme system, the bare data do not
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VOL. 34,VO.3,8 GENETICS: MAZIA AND BLUMENTHAL '33
permit a quantitative evaluation of the effect. For this purpose it would be
desirable to express the results as the proportion or number of active units
DOSe - ROENTGEN UMTS
FIGURE 1
Relation between diggstion rate and x-ray dose. Each point represents an
independent experiment. Ratios are albumin-pepsin ratios in solutions from which
films were prepared.
2 3 4 5
MINUTES TO DIGEST
FIGURE 2
Relation between enzyme content of films and digestion rate. Curve B represents
digestion rates for various albumin pepsin ratios (Scale B). Ratios are those in solu-
tions from which films were prepared. Curves A (read agqinst scale A) are calculated
from Curve B. Enzyme content of 20: 1 and 40:1 films are taken as unity, digestion
rate' is plotted against fractions of initial enzyme content.
affected. This is possible if a relation can be found between the number of
active units and the digestion rate. Such a "calibration" is obviously pos-
sible, for all that is necessary is to determine the digestion rate of films pre-
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pared from solutions containing various substrate enzyme ratios. The re-
sults of such a calibration are given in figure 2.
The use of this calibration depends on certain assumptions. First, it has
to be assumed that there is a definite relation between the enzyme and sub-
strate concentrations in the solutions and in the film. It is assumed, in the
plot, that the ratios in the film approximate those in the solutions. Second,
it must be assumed that any decrease in the digestion rate after irradiation
results from the inactivation of a certain proportion of enzyme molecules
rather than other conceivable causes. This assumption is difficult to test.
It is, however, present in every enzyme experiment, where the amount of
active enzyme is deduced by comparison with a concentration-activity
curve. The only alternative would be the impracticable one of isolating the
active fraction.
LO
40:1 20:1-
.6
0
h..4
.2
25 50 75 100 125 I50
DOSE ROCNTNEt& UNITS
FIGURE 3
Relation between x-ray dose and surviving active fraction of enzyme in film. Data
from figure 1 are replotted using calibration data of figure 2.
In figure 2, Curve B represents the actual relation between the substrate
enzyme ratio and the rate of digestion, as determined by separate experi-
ments on various mixtures. The other curves are based on the same data
and are introduced merely for convenience. They represent the relation be-
tween the active fraction of the enzyme and digestion rate where certain
substrate-enzyme ratios are taken as unity.
These curves may then be used to express the radiation data of figure 1 in
terms of the surviving fraction of the initial activity. Qbviously, they can
also be used to calculate the number of units inactivated, using the area of
the film and the molecular weights of the proteins.
Figure 3 shows the radiation data plotted as dose against active fraction.
If. the calibration of the method is valid, this plot establishes the high sen-
sitivity of the system in an absolute sense, for it is seen that a dose of 100 r
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destroys more than half of the activity present. Certain other points are
suggested by the plot.
1. The "threshold" effect suggested by figure 1 remains. The point of
deflection is difficult to determine as might be expected where individual
films vary in sensitivity.
2. There is an unexplained,difference between the two films of different
composition. The threshold effect is less pronounced in the film with a 40:1
substrate enzyme ratio than in the 20:1 film. Moreover the whole dose-
effect relationship is different for the two films. If we were dealing with a
"hit" relationship, we could expect th'e curves to coincide, since the fraction
affected would not be a -unction of the size of the population. If we were
dealing with an indirect action as described by Dale, it would be expected
60
s0
40 * 96e.
3O
z20,
I.
0o 20 *3b
PERCENT COMPRESSION
4:0 50
FIGURE 4
Relation between surface compression and radiation sensitivity. Compression
given in units of area, not pressure. Inactivation calculated from calibration in figure 2.
Curve represents inactivation by 96 r. The single point gives inactivation by 240 r
at 28 per cent compression.
that the total number of units affected by a given dose would be independ-
ent of the number present. This is not the case. It is obvious that the
composition affects the sensitivity in a way that is not simple.
Effect of Compression on Sensitivity.-These films are more sensitive to
radiation than would be predicted from their composition alone, and the
structural factors in their sensitivity have to be taken into account. One pos-
sibility is that the spreading of the enzyme molecule (however incomplete).
may increase its sensitivity. Another is that the structural continuity may
be a factor. These variables may be tested by determining the effect of
surface compression on radiation sensitivity. In these experiments, the
filmns were spread as already described, compressed to a given area, irradia-
ted, then fully compressed and tested for digestion rate. The results are
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given in figure 4. There is evidently a sharp, discontinuous effect. Sensi-
tivity begins to fall off at about 10 per cent compression, and at about 28
per cent compression is lowered to the point where the effects of a dose of 96
r are not detectable. Sensitivity is only Jowered, not destroyed. At 28 per
cent compression, 240 r produce about the same effect as 96 r at 13 per
cent compression. If area of exposure to irradiated medium determined the
sensitivity, one would expect some simple proportionality between area and
radiation effect instead of the discontinuous relationship observed. It
seems clear that, regardless of the mechanism of the radiation effect, the
effect is on the film itself rather than on dissolved enzyme and that the sen-
sitivity is determined by the physical configuration of the film. Obviously,
the "compression" shown in the data is only a very rough index of changes
in surface pressure, and it is necessary to investigate the relationship be-
tween actual pressure and sensitivity. Such an investigation is now under
way.
Calcuzations.-Although the experiments bearing on the mechanism of
x-ray action on the enzyme substrate films will be reported in a second
paper, the nature of the problem can be indicated by some pieliminary cal-
culations. In making these, we have relied on the tables compuited by Lea.6
First, the number of units affected may be estimated from figure 3. The
film area used in our experiments was 576 cm.2 Film pressure determina-
tions shoxy this to contain about 0.07 mg. of protein, or a total of 1015 mole-
cules of molecular weight 34,000. A 20:1 albumin pepsin film would con-
tain about 5 X 1013 pepsin molecules and a 40:1 film half this number. In
orders of magnitude, 100 r, accordinrg to figure 3, would produce inactiva-
tion equivalent to the removal of 101' pepsin molecules.
The expected number of ionizations in the film itself may be estimated
on the basis of the mass of protein, neglecting geometric considerations
which would make the true figure lower than the estimate. For the film
containing 0.07 mg. of protein, 100 r of 0.15 A x-rays would yield about
1010 ionizations, a figure lower by orders of magnitude than the estimated
number of pepsin molecules inactivated.
If the effect is through ionization products in the medium, the calculation
has to be made on the basis of unstable products of water decomposition.
Experiments in which films were spread over solution that had previously
received large doses of radiation showed no differences from controls, and
therefore the formation of adequate concentrations of stable products must
.be excluded. Using Lea's data again, it is calculated that the number of
radicals produced by 100 r is of the order of 1014 per cc. One may calculate
the time that would be required for 1013 radicals to reach the film if they
were stable. The equation for diffusion across a plane from a sen;i-infinite
column into a column of zero concentration is given by Jacobs 7 (equatibn
104). We use as diffusion constant the value of 2 X 10-5 cm.2 sec.-', the
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constant for -D20 in H20. The calculation shows that it requires 10-4-10-
seconds for 103 radicals to reach the surface. If, then, radicals produced by
the decomposition of water were the effective agents, the simple calculation
would require their half-life to be of this order. If only radicals finding a
pepsin molecule were effective, the time would be 20-40 times greater. Lea
estimates 10-7 seconds as their half-life in water, and less in the presence of
solutes such as citric acid which was present in 0.01 M concentration in our
experiments.
On the face.of these considerations, our results would require the assump-
tion that the effects of a single radiation event, whether in direct action
through ionization or indirect action through ionization produdts, are.
spread over a considerable number of pepsin molecules.8 These calculations
alone, however, cannot be considered to be a demonstration of the spread of
the effect. Lea's estimate of the half-life of radicals produced by irradiation
of water may be too low. Moreover, it may not be valid to base the number
of inactivations on numbers of pepsin molecules. The film may in fact be a
complex polymer, where the scope of a unit inactivation would be difficult
*to define.
Discussion.-We have restricted ourselves to the description of the radia-
tion sensitivity of the film enzyme-substrate system, and will present data
bearing on the mechanism of the effect in a subsequent publication. The
basic fact is that the pepsin-albumin films described are unexpectedly sensi-
tive to x-rays, doses of less than 200 r causing the maximum measurable
inactivation and doses of the order of 50 r producing readily detectable in-
activation. In absolute terms, this sensitivity exceeds that of biological
systems, where we do not obtain, with such doses, such large percentages of
all possible effects.
The dose-effect curves, with the indicated "threshold" effect, suggest
nothing so much as "multiple hit" curves, but their analysis must await the
decision as to the type of mechanism causing the effect.
The interest of these observations rests not so much on the sensitivity
itself, which might conceivably be duplicated in dilute pure solutions, as on
the fact that the sensitivity is-obtained in a situation conceptually compar-
able to that in chromosomes. By organizing the enzyme system into a solid
film we are liberated from the restrictions of a dilute solution. The films
are as "concentrated" as can be imagined and may contain any number of
components. The only possible "protective" action that need be considered
is that of the solutes in the adjacent liquid phase. Without pressing de-
tailed analogies between films and cellular structures, it is obvious that with
the films certain variables arising in cytological problems may be investi-
gated at a biochemical level. Examples are the question of the spreading
effect of a single radiation event within a structure, the range of ionization
products, and, particularly, the relation between physical structure and
Downloaded by guest on April 19, 2021336 3MA THEMA TICS: M. H. STONE PROC. N. A. S.
radiation sensitivity. The aim of further work along these lines is to find
ways of picturing biological effects of radiation and premises to be used in
their interpretation.
Summary.-(1) Pepsin-albumin films may be inactivated by small
doses of x-rays. Doses of 'about 100 r produce about 50 per cent inactiva-
tion.
(2) The sensitivity to radiation depends on the physical configuration
of the molecules. It may be varied by surface compression.
(3) Calculations suggest that the effects of a single ra.diation event
(ionization or radical production) may be spread to include a large
numbei of enzyme molecules.
* This work has been supported by a grant from the Committee on Growth, NATIONAL
RESBARCH COUNCIL, acting for the American Cancer Society. The authors wish to
thank Dr. A. C. Faberge for carrying out the dosimetry.
1 Mazia, D., Hayashi, T., and Yudowitch, K., Cold Spring Harbor Symp. Quant. Biol.,
12, 122 (1947).
2 La Rosa, W., Chemist Analyst, 12, 2 (1927).
3 Robinson, H. W., and Hogden, C. G., J. Biol. Cl'em., 135, 707 (1940).
4 Mazia, D., and Hayashi, T., unpublished.
6 Anson, M. L., J. Gen. Physiol., 22, 79 (1938-1939).
6 Lea, D. E., Action of Radiations on Living Cells, Cambridge, 1947.
7 Jacobs, M. H., Ergeb. d. Biol., 12, 1 (1935).
8 J. Weiss (Brit. J. Radiol., Suppl. No. 1, 56 (1947)) has recently proposed that a
radical produced by the ionization of solvent may initiate a chain reaction extending as
far as 10-' cm.
NOTES ON INTEGRATION, I
BY M. H. STONE
DEPARTMENT OF MATHEMATICS, THE UNIVERSITY OF CHICAGO
Communicated June 3, 1948
The theory of integration, because of its central rBle in mathematical
analysis and geometry, continues to afford opportunities for serious in-
vestigation. The need for extending and rounding out the classical
studies of Riemann, Stieltjes and Lebesgue has stimulated considerable
interest not only in new aspects of the theory but also in the simplification
and perfection of the old. The present series of communications is in-
tended to outline a treatment which, while exploiting fully the possibilities
for simplification, will attain 4 high degree of generality. Among the
ma.ny contributions to the mathematical literature which have provided
material for our handling of the subject we wish to cite above all an im-
portant paper of Daniell.- In spite of the fact that the basic ideas in the
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